Over the last several decades, mass spectrometry has emerged as one of the most broadly applicable analytical tools for detection and characterization of a wide class of molecules, ions and aggregates of molecules, ions or both. Mass spectrometric analysis is applicable to almost any species capable of forming an ion in the gas phase, and, therefore, provides perhaps the most universally applicable method of quantitative analysis. In addition, mass spectrometry is a highly selective technique especially well suited for the analysis of complex mixtures comprising a large number of different compounds in widely varying concentrations. Moreover, mass spectrometric methods provide very high detection sensitivity, approaching tenths of parts per trillion for some species.
As a result of the universal, selective and sensitive detection provided by mass spectrometry, a great deal of attention has been directed at developing mass spectrometric methods for analyzing complex mixtures of biomolecules. Indeed, the ability to efficiently detect components of complex mixtures of biological compounds via mass spectrometry would aid tremendously in the advancement of several important fields of scientific research. First, advances in the characterization and detection of samples containing mixtures of oligonucleotides by mass spectrometry would improve the accuracy, speed and reproducibility of DNA sequencing methodologies. Such advances would also eliminate problematic interference arising from secondary structure, which can be observed in conventional gel electrophoresis sequencing methodologies. Second, enhanced capability for the analysis of complex protein mixtures and multi-subunit protein complexes would revolutionize the use of mass spectrometry in proteomics. Important applications of mass spectrometry to proteomics include: protein identification, relative quantification of protein expression levels, single cell analysis, identification of protein post-translational modifications, and the analysis of labile protein—protein, protein—DNA and protein—small molecule aggregates. Finally, advances in mass spectrometric analysis of samples comprising complex mixtures of biomolecules would also allow the simultaneous characterization of high molecular weight and low molecular weight compounds. Detection and characterization of low molecular weight compounds, such as glucose, ATP, NADH, GHT, would aid considerably in elucidating the role of these molecules in regulating important cellular processes. While the benefits of mass spectrometric techniques for the analysis of complex mixtures of biological compounds are clear, the full potential for quantitative analysis of biological samples remains unrealized because there remain substantial problems in producing, analyzing and detecting gas phase ions generated from high molecular weight compounds.
Mass spectrometric analysis involves three fundamental processes: (1) gas phase ion formation, (2) mass analysis whereby ions are separated on the basis of mass-to-charge ratio (m/z) and (3) detection of ions subsequent to their eparation. The overall efficiency of a mass spectrometer (overall efficiency=(analyte ions detected)/(analyte molecules consumed)) may be defined in terms of the efficiencies of each of these fundamental processes by the equation:EMS=EF×EMA×ED,  (I)where EMS is the overall efficiency, EF is the ion formation efficiency (ion formation efficiency=(analyte ions formed)/(analyte molecules consumed during ion formation)), EMA is the mass analysis efficiency (mass analysis efficiency=(analyte ions mass analyzed)/(analyte ions consumed during analysis)) and ED is the detection efficiency (detection efficiency=(analyte ions detected)/(analyte ions consumed during detection)). Although mass spectrometry has been demonstrated to provide an important means of identifying biomolecules, current mass spectrometers have surprisingly low overall efficiencies for these compounds. For example, a quantitative evaluation of the efficiency of a conventional orthogonal injection time-of-flight mass spectrometer (Perseptive Biosystems Mariner) for the analysis of a sample containing a 10 kDa protein yields the following efficiencies, ES=1×10−4, EMA=8×10−7, and ED=9×10−3, providing an overall efficiency of the mass spectrometer of 1 part in 1012. As a result of low overall efficiency, conventional mass spectrometric analysis of biomolecules requires larger quantities of biological samples and is unable to achieve the ultra low sensitivity needed for many important biological applications, such as single cell analysis of protein expression and post-translational modification. Therefore, there is a significant need in the art for more efficient ion preparation, analysis and detection techniques to capture the full benefit of mass spectrometric analysis for important biological applications.
Over the last decade, new ion preparation methods have been developed, such as matrix assisted laser desorption and ionization (MALDI) and electrospray ionization (ESI). These ionization methods provide greatly improved ionization efficiency for a wide range of compounds having molecular weights up to several hundred kiloDaltons. Moreover, MALDI and ESI ionization sources have been successfully coupled to a variety of mass analyzers, including quadrupole mass analyzers, time-of-flight instrumentation, magnetic sector analyzers, Fourier transform—ion cyclotron resonance instruments and ion traps, to provide selective identification of polypeptides and oligonucleotides in complex mixture of biological compounds. Mass analysis by orthogonal time-of-flight (TOF) methods has proven especially compatible for the analysis of high molecular weight biomolecules because they have no intrinsic limit to the mass range accessible, provides high spectral resolution and has a fast temporal response. The use of time-of-flight mass analysis with ESI and MALDI ion sources for proteomic analysis is described by Yates in Mass Spectrometry and the Age of the Proteome, Journal of Mass Spectrometry, Vol. 33, 1–19 (1998). As a result, MALDI-TOF and ESI-TOF have emerged as the two most commonly used mass spectrometric techniques for analyzing complex mixtures of biomolecules having high molecular weight.
In MALDI-TOF mass spectrometry, an analyte of interest is co-crystallized with a small organic compound present in high molar excess relative to the analyte, called the matrix. The MALDI sample, containing analyte incorporated into the organic matrix, is irradiated by a short (≈10 ns) pulse of UV laser radiation at a wavelength resonant with the absorption band of the matrix molecules. Rapid absorption of energy by the matrix causes it to desorb into the gas phase, thereby, volatilizing a portion of the analyte molecules. Gas phase proton transfer reactions ionize the analyte molecules within the resultant gas phase plume and generate gas phase analyte ions in singly and/or multiply charged states. Ions in the source region are accelerated by a high potential electric field, which imparts equal kinetic energy to each ion, and are conducted through an electric field-free flight tube. The ions are separated according to their velocities and are detected by a detector positioned at the end of the flight tube. Accordingly, light ions having higher velocities reach the detector first, while heavier ions having lower velocities arrive later.
In ESI-TOF mass spectrometry, a solution containing solvent and analyte is passed through a capillary orifice and directed at an opposing plate held near ground. The capillary is maintained at a substantial electric potential (approximately 4 kV) relative to the opposing plate, which serves as the counter electrode. This potential difference generates an intense electric field at the capillary tip, which draws some free ions in the exposed solution to the surface. The electrohydrodynamics of the charged liquid surface causes it to form a cone, referred to as a “Taylor cone.” A thin filament of solution extends from this cone until it breaks up into droplets, which carry excess charge on their surface. The result is a stream, of small, highly charged droplets that migrate toward the grounded plate. Facilitated by heat, the flow of dry bath gases or both, solvent from the droplets evaporates and the physical size of the droplets decreases to a point where the force due to repulsion of the like charges contained on the surface overcomes surface tension and causes the droplets to fission into “daughter droplets.” This fissioning process may repeat several times depending on the initial size of the parent droplet. Eventually, daughter droplets are formed with a radius of curvature small enough that the electric field at their surface is large enough to desorb analyte species existing as ions in solution. Polar analyte species may also undergo desorption and ionization during electrospray by associating with cations and anions in the liquid sample. Further, analyte ions may be formed from substantially complete desolvation of solvent from the charged droplets. The electrospray-generated ions are periodically pulsed into an electric field-free-flight tube positioned orthogonal to the axis along which the ions are generated. Ideally, all ions having the same charge-state are imparted with the same kinetic energy and, therefore, analyte ions in the flight tube are separate by mass according to their velocity. Lighter ions translate at higher velocities and are detected earlier in time by an ion detector positioned at the end of the flight tube, while heavier ions translate at lower velocities and are detected later in time.
Although the combination of modern ionization techniques and time-of-flight analysis methods has greatly expanded the mass range accessible by mass spectrometric methods, complementary ion detection methods suitable for the time of flight analysis of high molecular weight compounds remain less well developed. Indeed, the effective upper limit of mass ranges currently accessible by MALDI-TOF and ESI-TOF analysis techniques are limited by the sensitivity of conventional ion detectors for high molecular weight ions. For example, multichannel plate (MCP) detectors exhibit detection sensitivities that decrease with ion velocity. In time-of-flight analysis, this corresponds to a decrease in sensitivity with increasing molecular weight.
MCP detectors are perhaps the most pervasive ion detector in ESI-TOF and MALDI-TOF mass spectrometry. These detectors operate by secondary electron emission. Specifically, MCP detectors comprise a plurality of MCP channels, each of which release secondary electrons upon collision of a gas phase ion with a channel surface. Ejected secondary electrons are subsequently accelerated down discrete MCP channels and generate additional secondary electrons upon further collisions with the walls of the MCP channel. The electron cascade formed is collected at an anode and generates an output signal.
A number of substantial limitations of this detection technique arise out of the impact-induced mechanism of MCP detectors governing secondary electron generation. First, the yield of secondary electrons in a MCP detector decreases significantly as the velocity of ions colliding with the surface decreases. As time-of-flight detectors accelerate all ions to a fixed kinetic energy, high molecular weight ions have lower velocities and, hence, lower probabilities of being detected by MCP detectors. Second, the secondary electron yield of MCP detectors also depends on the composition and structure of colliding gas phase ions. Third, MCP detection is a destructive technique incapable of detecting the same ion or packet ions multiple times. Finally, MCP detectors generate electron cascades upon the impact of any species with the channel surface, including unwanted neutral species present in the ion flight tube.
As is apparent to those skilled in the art of mass spectrometry, the limitations associated with MCP detectors restrict the mass range currently accessible by MALDI-TOF and ESI TOF techniques, and hinder the quantitative analysis of samples containing high molecular weight biopolymers. Accordingly, there currently exists a need for ion detectors that do not exhibit decreasing sensitivities with increasing molecular weight and that do not have sensitivities dependent on the composition and structure of gas phase ions analyzed.
Over the last decade, considerable research has been directed at developing new ion detectors suitable for high molecular weight compounds. For example, inductive detectors have been developed that provide a non-destructive means of detecting highly multiply charged ions having high molecular weights. Park and Callahan, Rapid Comm. Mass Spec., 8, 317–322 (1988), Lennon et al., Anal. Chem., 68, 845–849 (1996), and Benner, Anal. Chem., 69, 4162–4168 (1997) describe applications of inductive detectors in mass spectrometric analysis. Inductive detectors operate by generating an induced electric charge upon interaction of gas phase ions with the surface of a sensing electrode. A primary advantage of inductive detectors is that they are sensitive only to an ion's charge, not an ion's velocity. In addition, inductive detectors are non-destructive. Therefore, a series of inductive detectors is capable of providing multiple detection methods wherein an ion or ion packet is repeatedly analyzed and detected. Although inductive detectors have been successfully applied to Fourier transform mass spectrometry, their use in time-of-flight mass analysis is substantially limited due to low sensitivity and poor detection efficiency.
U.S. Pat. No. 5,591,969 discloses a single inductive detector comprising a sensing tube providing non-destructive, time-of-flight analysis of ion packets. The cylindrical sensing electrode is configured to generate an induced electric charge upon passage of gas phase analyte ions through an axial bore in the detector. Although the detector reportedly provides detection sensitivity that is independent of velocity, the single electrode arrangement does not provide a means of characterizing the velocities of ions prior to acceleration and time-of-flight analysis. This limitation substantially reduces the mass resolution of the disclosed detector. In addition, the methods and devices described are limited to detection of packets of gas phase ions, rather than single ions. Finally, U.S. Pat. No. 5,591,969 is limited to embodiments employ a relatively short ion flight path corresponding to the length of a short sensing tube.
U.S. Pat. No. 5,770,857 discloses a method and apparatus for determining molecular weight which combines conventional ESI ion formation methods and an ion detection scheme comprising a first cylindrical inductive detector positioned a selected distance upstream of a second ion detector. The inductive detector is configured to provide a measurement of the start time of gas phase ions translating a flight path from first inductive detector to the second detector. Although U.S. Pat. No. 5,770,857 describes analysis methods employing a series of two detectors, the detector arrangement is reported to provide very low ion transmission efficiencies from an ion formation region to ion analysis and detection regions. Further, the mass analysis method of U.S. Pat. No. 5,770,857 relies on estimates of pre-acceleration ion velocity rather than direct measurements or ion velocity. Because knowledge of pre-acceleration ion velocity is critical for the accurate determination of mass-to-charge ratio, uncertainty in this important parameter degrades mass resolution and absolute mass accuracy attainable. Moreover, the spatial distribution of ions generated by the ion source and transmission scheme of the disclosed method substantially limits the sensitivity, mass analysis efficiency and detection efficiency attainable. First, free expansion of ions prior to detection results in a wide spatial distribution of gas phase ions. This spatial distribution reflects a wide variation in ion trajectories through the time-of-flight mass separation region, which substantially limits the diameters and lengths of cylindrical ion detectors employable. Second, the spatial distribution of the ions sampled impedes effective use of multiple inductive detectors in series because ion trajectories, which deviate substantially from the centerline of the detection scheme, will not be efficiently sampled by detectors positioned toward the end of a long flight path (>1 meter). Finally, the detection technique described provides a relatively low detection sensitivity, limited to detecting ions having charge states of hundreds of elemental charges.
It will be appreciated from the foregoing that a need exists for methods and devices suitable for efficient and sensitive analysis and detection of high molecular weight ions. Particularly, ion detectors having a detection sensitivity independent of molecular mass and structure are needed. Accordingly, it is an object of the present invention to provide methods, devices and device components capable of efficient analysis and detection of high molecular weight ions having high masses, particularly biomolecules. The present invention provides improved methods and devices for time-of-flight analysis combining spatially collimate electrically charged particle sources and multiple, non-destructive inductive detection. The analysis and detection methods of the present invention provide direct measurement of pre-acceleration and post-acceleration velocities and are capable of diverse applications of electrically charged particle analysis in coincidence, which substantially improves the sensitivity, resolution and absolute mass accuracy of time-of-flight analysis of high molecular weight ions.